Method of detecting β3 adrenaline receptor mutant gene and nucleic acid probe and kit therefor

ABSTRACT

A melting curve analysis is performed for a nucleic acid containing a mutation in a nucleotide sequence resulting in a mutation replacing tryptophan at position 64 in an amino acid sequence of the β3-adrenergic receptor with arginine (B3AR Trp64Arg), by using a nucleic acid probe of which end is labeled with a fluorescent dye, and in which fluorescence of the fluorescent dye decreases upon hybridization, wherein the nucleic acid probe has a nucleotide sequence starting from the nucleotide number 183 in the nucleotide sequence of SEQ ID NO: 1 and having a length of 8 to 30 nucleotides, and the 5′ end of the probe is labeled with the fluorescent dye, or the nucleic acid probe has a nucleotide sequence ending at the nucleotide number 196 in the nucleotide sequence of SEQ ID NO: 2 and having a length of 7 to 30 nucleotides, and the 3′ end of the probe is labeled with the fluorescent dye, and measuring fluorescence of the fluorescent dye, and the mutation is detected on the basis of the result of the melting curve analysis.

RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication PCT/JP2004/005525, filed Apr. 16, 2004, which was publishedin a language other than English which claims priority of JP2003-114381, filed Apr. 18, 2003.

TECHNICAL FIELD

The present invention relates to method for detecting a β3-adrenergicreceptor mutant gene, and a nucleic acid probe and a kit therefor.

BACKGROUND ART

The β3-adrenergic receptor (B3AR) plays a major part in lypolysis inwhite adipose cells and heat generation in brown adipose cells. Theexistence of the mutation replacing tryptophan at position 64 in theamino acid sequence of B3AR with arginine (Trp64Arg) is said to reducethe resting metabolic rate by 200 kcal, and to be involved in obesitywith abdominal fat and in insulin resistance.

If the mutation resulting in the Trp64Arg mutation in B3AR (alsoreferred to as “B3AR Trp64Arg mutation”) exists, a recognition site of arestriction enzyme emerges at the position of the mutation. Therefore,the mutation can be detected by a method of amplifying DNA by PCR sothat a portion including the mutation position should be amplified,digesting the amplification product with a restriction enzyme anddetermining whether the DNA has been digested or not by electrophoresis(PCR-RFLP) (for example, refer to The Japanese Journal of ClinicalPathology, vol. 44, 8, pp. 778-782, 1996).

Because PCR amplifies templates of several molecules several billiontimes, even a trace amount of contaminant may cause a false positive orfalse negative result. In PCR-RFLP, the amplification product needs tobe collected and subjected to a treatment with a restriction enzymeafter PCR, and therefore the amplification product may contaminate thesubsequent reaction system. Accordingly, a false positive or falsenegative result may be obtained.

Further, DNA is treated with a restriction enzyme and then subjected toelectrophoresis after completion of PCR. Therefore, time required forthe detection becomes extremely long. In addition, because the procedureis complicated, automatization is difficult.

Furthermore, a method is generally known in which a region containing amutation is amplified by PCR, then a melting curve analysis is performedby using a nucleic acid probe labeled with a fluorescent dye, and themutation is analyzed on the basis of the result of the melting curveanalysis (Clinical Chemistry, vol. 46, 5, pp. 631-635, 2000; JapanesePatent Application Laid-open (Kokai) No. 2002-119291).

DISCLOSURE OF THE INVENTION

An object of the present invention is to identify a quenching probeeffective for the detection of the B3AR Trp64Arg mutation and therebyprovide a method for detecting the B3AR Trp64Arg mutation and a kittherefor.

The literature concerning the aforementioned method of using a probeonly teaches that, concerning the design of the probe, the probe shouldbe designed so that, when a quenching probe having an end labeled with afluorescent dye hybridizes with a target nucleic acid, two or morenucleotide pairs of the probe-nucleic acid hybrid should form at leastone pair of G and C in the end portion. With regard to the B3AR Trp64Argmutation, the inventors of the present invention designed a quenchingprobe satisfying the aforementioned condition and attempted thedetection. However, no quenching probe that enabled detection was easilyobtained.

The inventors of the present invention found that by designing aquenching probe based on a specific region containing the B3AR Trp64Argmutation, the B3AR Trp64Arg mutation could be detected by a meltingcurve analysis using the quenching probe.

The present invention provides the followings.

(1) A nucleic acid probe of which end is labeled with a fluorescent dye,and in which fluorescence of the fluorescent dye decreases uponhybridization, wherein the nucleic acid probe has a nucleotide sequencestarting from the nucleotide number 183 in the nucleotide sequence ofSEQ ID NO: 1 and having a length of 8 to 30 nucleotides, and the 5′ endof the probe is labeled with the fluorescent dye, or the nucleic acidprobe has a nucleotide sequence ending at the nucleotide number 196 inthe nucleotide sequence of SEQ ID NO: 2 and having a length of 7 to 30nucleotides, and the 3′ end of the probe is labeled with the fluorescentdye.(2) The nucleic acid probe according to (1), wherein the nucleic acidprobe has any one of the nucleotide sequences of SEQ ID NOS: 8 to 12.(3) A method for detecting a mutation comprising performing a meltingcurve analysis for a nucleic acid having a single nucleotidepolymorphism site by using a nucleic acid probe labeled with afluorescent dye and measuring fluorescence of the fluorescent dye, anddetecting the mutation on the basis of the result of the melting curveanalysis, wherein the single nucleotide polymorphism is a mutation in anucleotide sequence in a nucleic acid encoding a β3-adrenergic receptor,resulting in a mutation replacing tryptophan at position 64 in an aminoacid sequence of the β3-adrenergic receptor with arginine, and thenucleic acid probe is the nucleic acid probe as defined in (1) or (2).(4) The method according to (3), wherein a region containing a singlenucleotide polymorphism site in a nucleic acid contained in a sample isamplified to obtain the nucleic acid showing the single nucleotidepolymorphism.(5) The method according to (4), wherein the amplification is performedby a method of using a DNA polymerase.(6) The method according to (5), wherein the amplification is performedin the presence of a nucleic acid probe.(7) A kit for the method as defined in (3), which comprises a nucleicacid probe of which end is labeled with a fluorescent dye, and in whichfluorescence of the fluorescent dye decreases upon hybridization,wherein the nucleic acid probe has a nucleotide sequence starting fromthe nucleotide number 183 in the nucleotide sequence of SEQ ID NO: 1 andhaving a length of 8 to 30 nucleotides, and the 5′ end of the probe islabeled with the fluorescent dye, or the nucleic acid probe has anucleotide sequence ending at the nucleotide number 196 in thenucleotide sequence of SEQ ID NO: 2 and having a length of 7 to 30nucleotides, and the 3′ end of the probe is labeled with the fluorescentdye.(8) The kit according to (7), wherein the nucleic acid probe has any oneof the nucleotide sequences of SEQ ID NOS: 8 to 12.(9) The kit according to (7) or (8), which further comprises a primerfor amplifying a region containing a mutation in a nucleotide sequencein a nucleic acid encoding a β3-adrenergic receptor, resulting in amutation replacing tryptophan at position 64 in an amino acid sequenceof the β3-adrenergic receptor with arginine, by a method of using a DNApolymerase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows positions of quenching probes that cannot identify amutation.

FIG. 2 shows positions of quenching probes that can identify a mutation.

FIG. 3 shows sensitivity of the method of Example 1 (using probe5FL-wt-1-16) with respect to the absolute amount of genomic DNA.

FIG. 4 shows reproducibility of the method of Example 1 (using probe5FL-wt-1-16).

FIG. 5 shows sensitivity of the method of Example 1 (using probe3T-mt-2-20) with respect to the absolute amount of genomic DNA.

FIG. 6 shows reproducibility of the method of Example 1 (using3T-mt-2-20).

BEST MODE FOR CARRYING OUT THE INVENTION

<1> Probe of the Present Invention and Detection Method of the PresentInvention

The probe of the present invention is a nucleic acid probe of which endis labeled with a fluorescent dye, and in which fluorescence of thefluorescent dye decreases upon hybridization, wherein the probe has anucleotide sequence starting from the nucleotide number 183 in thenucleotide sequence of SEQ ID NO: 1 and having a length of 8 to 30nucleotides, and the 5′ end of the probe is labeled with the fluorescentdye, or the probe has a nucleotide sequence ending at the nucleotidenumber 196 in the nucleotide sequence of SEQ ID NO: 2 and having alength of 7 to 30 nucleotides, and the 3′ end of the probe is labeledwith the fluorescent dye.

The probe of the present invention may be similar to the quenching probedescribed in Patent Document 1 except that it has a nucleotide sequencestarting from the nucleotide number 183 in the nucleotide sequence ofSEQ ID NO: 1 (sequence having the wild type nucleotide in the B3ARTrp64Arg mutation) and having a length of 8 to 30 nucleotides, or anucleotide sequence ending at the nucleotide number 196 in thenucleotide sequence of SEQ ID NO: 2 (sequence having the mutant typenucleotide in the B3AR Trp64Arg mutation) and having a length of 7 to 30nucleotides. Examples of the nucleotide sequence of the quenching probeused in the present invention include the nucleotide sequences of SEQ IDNOS: 8 to 12. As the fluorescent dye, those described in Patent Document1 can be used, and specific examples thereof include FAM (trademark),TAMRA (trademark), BODIPY (trademark) FL and so forth. The fluorescentdye can be bound to an oligonucleotide in an ordinary manner, forexample, by the method described in Patent Document 1.

The detection method of the present invention is a method for detectinga mutation by performing a melting curve analysis for a nucleic acidhaving a single nucleotide polymorphism site by using a nucleic acidprobe labeled with a fluorescent dye and measuring fluorescence of thefluorescent dye, and detecting the mutation on the basis of the resultof the melting curve analysis, and characterized in that the singlenucleotide polymorphism is the B3AR Trp64Arg mutation, and the nucleicacid probe is the probe of the present invention.

The detection method of the present invention can be performed accordingto usual methods for nucleic acid amplification and melting curveanalysis (Tm analysis) except that a region containing the B3AR Trp64Argmutation in a DNA encoding B3AR is amplified, and the probe of thepresent invention is used.

As the method for nucleic acid amplification, a method of using apolymerase is preferred, and examples thereof include PCR, ICAN, LAMPand so forth. When amplification is performed by a method using apolymerase, amplification is preferably performed in the presence of theprobe of the present invention. The reaction conditions of theamplification and others can be easily adjusted depending on the usedprobe by those skilled in the art. In this method, only Tm of the probeis analyzed after amplification of a nucleic acid, and therefore it isnot necessary to handle the amplification product after completion ofthe reaction. Thus, there is no risk of contamination with theamplification product. Further, because the detection is performed withthe same equipment as required for the amplification, it is not evennecessary to move a vessel. Therefore, automatization of the method isalso easy.

The method will be further explained below by referring, as an example,to a case of using PCR. The primer pair used for PCR can be designed inthe same manner as in a method for designing a primer pair in usual PCRexcept that it is designed so that a region to which the probe of thepresent invention is hybridizable should be amplified. The length and Tmof the primers are usually 10- to 40-mer and 40 to 70° C., preferably15- to 25-mer and 55 to 60° C., respectively. Primers of the primer pairmay not be equal in length. However, it is preferred that the Tm valuesof the primers are substantially equal (the difference is usually within2° C.). The Tm values are values calculated by the nearest neighbormethod. Examples of the primer pair include a primer pair comprisingprimers having the nucleotide sequences of SEQ ID NOS: 3 and 4.

PCR is preferably performed in the presence of the probe of the presentinvention. This enables the Tm analysis without performing any operationof handling the amplification product after completion of theamplification reaction. Tm values of primers and reaction conditions ofPCR can be easily adjusted by those skilled in the art depending on theused probe.

A typical example of the composition of the reaction mixture for PCR isas follows.

TABLE 1 DNA fragments 10¹ to 10⁸ molecules/reaction Primers 200 to 1000nM Probe 100 to 1000 nM Nucleotides 20 to 200 μM each DNA polymerase0.01 to 0.03 U/μl Tris-HCl (pH 8.4 to 9.0) 5 to 20 mM MgCl₂ 1.5 to 3 mMKCl 10 to 100 mM Glycerol 0 to 20% (Final fluid volume: 10 to 100 μl)

Further, a typical example of the temperature cycle is as follows, andthis temperature cycle is usually repeated 25 to 40 times.

(1) Denaturation at 90 to 98° C. for 1 to 60 seconds

(2) Annealing at 60 to 70° C. for 10 to 60 seconds

(3) Extension at 60 to 75° C. for 10 to 180 seconds

When annealing and extension are performed in one step, conditions of 60to 70° C. for 10 to 180 seconds can be mentioned, for example.

The Tm analysis can be performed in a conventional manner except thatfluorescence of the fluorescent dye binding to the probe of the presentinvention is measured. Fluorescence can be measured by using excitationlight having a wavelength suitable for the fluorescent dye and measuringintensity of light of the emission wavelength. The temperatureincreasing rate in the Tm analysis is usually 0.1 to 1° C. per second.Composition of the reaction mixture for Tm analysis is not particularlylimited so long as a probe and a nucleic acid having a sequencecomplementary to the nucleotide sequence of the prove can hybridize toeach other. However, the monovalent cation concentration is usually 1.5to 5 mM, and pH is usually 7 to 9. Because a reaction mixture for anamplification method using a DNA polymerase such as PCR usuallysatisfies these conditions, the reaction mixture after the amplificationcan be used as it is for the Tm analysis.

The B3AR Trp64Arg mutation can be detected on the basis of the resultsof the Tm analysis in an ordinary manner. The detection in the detectionmethod of the present invention include not only detection of thepresence or absence of a mutation, but also quantification of mutanttype DNA and determination of the ratio of wild type DNA and mutant typeDNA.

<2> Kit of the Present Invention

The kit of the present invention is a kit used for the second detectionmethod of the present invention. This kit is characterized by includinga nucleic acid probe of which end is labeled with a fluorescent dye andin which fluorescence of the fluorescent dye decreases uponhybridization (quenching probe), wherein the nucleic acid probe has anucleotide sequence starting from the nucleotide number 183 in thenucleotide sequence of SEQ ID NO: 1 and having a length of 8 to 30nucleotides, and the 5′ end of the probe is labeled with the fluorescentdye, or the nucleic acid probe has a nucleotide sequence ending at thenucleotide number 196 in the nucleotide sequence of SEQ ID NO: 2 andhaving a length of 7 to 30 nucleotides, and the 3′ end of the probe islabeled with the fluorescent dye.

The quenching probe is as explained above with regard to the probe ofthe present invention.

The kit of the present invention may include reagents required foramplification of a nucleic acid in the detection method of the presentinvention, in particular, primers for amplification using a DNApolymerase, in addition to the quenching probe.

In the kit of the present invention, the quenching probe, primers andother reagents may be separately included, or a part thereof may beprovided as a mixture.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples.

Example 1

The primers shown in Table 2 were designed on the basis of thenucleotide sequence containing the site of the Trp64Arg mutation ofhuman B3AR gene (SEQ ID NO: 1) so that a region containing the Trp64Argmutation could be amplified. In Table 2, the positions are indicatedwith the nucleotide numbers in the nucleotide sequence of SEQ ID NO: 1.

TABLE 2 Primers SEQ ID Name Sequence (5′ → 3′) mer Position NO: Rgccagcgaagtcacgaacac 20 239-220 3 F Ggcgctggcggtgc 14 132-145 4

Then, the probes having C at the ends shown in Table 3 were designed. InTable 3, the positions are indicated with the nucleotide numbers in thenucleotide sequence of SEQ ID NO: 1. Further, the capital letters in thenucleotide sequences represent sites of the B3AR Trp64Arg mutation, and(P) at the 3′ ends means being phosphorylated. The probes were labeledwith BODIPY (trademark) FL or TAMRA (trademark) in a conventionalmanner.

TABLE 3 Probes SEQ ID Name Sequence (5′ → 3′) Mer Position NO:5FL-mt-4-16 (BODIFY FL)-ccatcgccCggactcc-(P) 16 182-197 5 3T-mt-4-16ccatcgccCggactcc-(TAMRA) 16 182-197 5 5FL-mt-4-19 (BODIPYFL)-ccatcgccCggactccgag-(P) 19 182-200 6 3T-mt-3-19gtcatcgtggccatcgccC-(TAMRA) 19 172-190 7 3T-mt-2-20cgtggccatcgccCggactc-(TAMRA) 20 177-196 8 5FL-wt-1-20 (BODIPYFL)-catcgccTggactccgagac-(P) 20 183-202 9 5FL-wt-1-18 (BODIPYFL)-catcgccTggactccgag-(P) 18 183-200 10 5FL-wt-1-16 (BODIPYFL)-catcgccTggactccg-(P) 16 183-198 11 5FL-wt-1-15 (BODIPYFL)-catcgccTggactcc-(P) 15 183-197 12

PCR and Tm analysis were performed by using genomic DNA as a sample andSmart Cycler System (Cephied) under the conditions shown below. Theexcitation wavelength and the detection wavelength in the Tm analysiswere 450 to 495 nm and 505 to 537 nm (BODIPY FL) and 527 to 555 nm and565 to 605 nm (TAMRA), respectively.

TABLE 4 Composition of reaction mixture H₂O 13.2 μL 10× Gene Taq buffer2.5 μL 80% Glycerol 6.25 μL 10 mM each dATP, dUTP, dGTP, dCTP 0.5 μL 2U/μL Uracil-N-glycosylase 0.05 μL 5 μM Probe 1 μL 100 μM Primer F 0.125μL 100 μM Primer R 0.25 μL 5 U/μL Gene Taq FP 0.125 μL Sample (0 to 2000copies) 1 μL Total 25 μL

TABLE 5 Reaction conditions

(50 cycles)

PCR and Tm analysis were performed by using each probe. As a result,only when the probes 3T-mt-2-20, 5FL-wt-1-20, 5FL-wt-1-18, 5FL-wt-1-16and 5FL-wt-1-15 were used, changes in fluorescence intensity that couldbe analyzed in Tm analysis were observed. The positions of the probesrelative to the nucleotide sequence containing the B3AR Trp64Argmutation are shown in FIGS. 1 and 2. The wild type sequence and mutanttype sequence shown in the drawings correspond to the nucleotide numbers171 to 205 in the nucleotide sequences of SEQ ID NOS: 1 and 2,respectively. Further, in the drawings, F denotes a fluorescent dye. Onthe basis of the positions shown in FIGS. 1 and 2, it is considered thatwhether the probe can be used for Tm analysis depends on the position ofC bound with a fluorescent dye, and the length of the probe is not soimportant so long as the polymorphism site is included.

In the following, by using the probe 5FL-wt-1-16, sensitivity withrespect to the absolute amount of genomic DNA, and reproducibility wereexamined.

The above method was repeated by using samples containing 0, 20, 200 and2000 copies of genomic DNA (wild type). The results are shown in FIG. 3.As seen from FIG. 3, it is shown that the genomic DNA can be detectedeven if 20 copies.

Then, plasmid having the wild type sequence (same as the above plasmidexcept that nucleotide number 285 is A in the nucleotide sequence of SEQID NO: 1) was prepared. Ten samples (wt/mt) were prepared by mixing thewild type plasmid and the mutant type plasmid. With respect to each ofthese samples as well as a sample (wt/wt) of only the wild type plasmidand a sample (mt/mt) of only the mutant type plasmid, the above methodwas repeated. The results are shown in FIG. 4. As seen from FIG. 4, itis shown that the method is excellent in the reproducibility.

Furthermore, by using the probe 3T-mt-2-20 instead of the probe5FL-wt-1-16, sensitivity with respect to the absolute amount of genomicDNA, and reproducibility were examined in the similar manner. Theresults are shown in FIGS. 5 and 6. As seen from FIGS. 5 and 6, it isshown that the sensitivity is high and the reproducibility is excellent.

In FIGS. 3 to 6, the vertical axis represents a primary derivative valueof fluorescence intensity with an inverted sign (−dF/dt), and thehorizontal axis represents temperature (° C.).

INDUSTRIAL APPLICABILITY

According to the present invention, a quenching probe effective fordetecting the B3AR Trp64Arg mutation is provided, and a method fordetecting the B3AR Trp64Arg mutation by using it and a kit therefor arefurther provided. Because the Tm analysis is completed within severaltens of seconds, time required for the detection can be markedlyreduced. According to a preferred embodiment of the present invention,wherein amplification of nucleic acid in the presence of the probe andTm analysis are combined, only the Tm of the probe is analyzed after theamplification of nucleic acid, and therefore it is not necessary tohandle the amplification product after completion of the reaction.Accordingly, there is no risk of contamination with the amplificationproduct. Further, because the detection can be performed with the sameequipment as required for the amplification, it is even unnecessary tomove a vessel. Therefore, automatization of the method is also easy.

1. A nucleic acid probe consisting of a nucleotide sequence ending atthe nucleotide number 196 in the nucleotide sequence of SEQ ID NO: 2 andhaving a length of 14 to 30 nucleotides, wherein position 196 of theprobe is cytosine labeled with a fluorescent dye.
 2. The nucleic acidprobe according to claim 1, wherein the nucleic acid probe has thenucleotide sequence of SEQ ID NO:
 8. 3. A method for detecting amutation comprising hybridizing a probe consisting of a nucleotidesequence ending at the nucleotide number 196 in the nucleotide sequenceof SEQ ID NO: 2 and having a length of 14 to 30 nucleotides with anucleic acid having a single nucleotide polymorphism site at position 64in the amino acid sequence of the β3-adrenergic receptor, whereinposition 196 of the probe is cytosine labeled with a fluorescent dye,performing a melting curve analysis, measuring fluorescence of thefluorescent dye, and detecting the mutation on the basis of the resultof the melting curve analysis, wherein the single nucleotidepolymorphism is a mutation in a polynucleotide encoding theβ3-adrenergic receptor, resulting in a mutation replacing tryptophan atposition 64 in the amino acid sequence of the β3-adrenergic receptorwith arginine and wherein the method is capable of detecting copy numberas low as 20 genomic copies.
 4. The method according to claim 3, whereina region containing the single nucleotide polymorphism site in apolynucleotide contained in a sample is amplified to obtain the nucleicacid showing the single nucleotide polymorphism.
 5. The method accordingto claim 4, wherein the amplification is performed using a DNApolymerase.
 6. The method according to claim 5, wherein theamplification is performed in the presence of a nucleic acid probe.
 7. Akit comprising a nucleic acid probe according to claim
 1. 8. The kitaccording to claim 7, wherein the nucleic acid probe has the nucleotidesequence of SEQ ID NO:
 8. 9. The kit according to claim 7, which furthercomprises a primer.